The long term objective of this renewal application is to develop therapeutic Na+ channel blockers pertinent to pain management. Traditional local anesthetics are often unsuited for treatment of chronic or intractable cancer pain because of their insufficient duration of nerve block. There are 4 specific aims: 1. To study the structure-activity relationship of various potent Na+ channel blockers in vitro; 2. To design and synthesize their amphipathic derivatives; 3. To test selected blockers suitable for prolonged nerve block in vivo; and 4. To map their receptor site within the Na+ channel alpha subunit. The first agent to be tested is the tricyclic antidepressant amitryptyline, which is a potent sodium channel blocking agent in addition to its actions at other receptors. With bupivacaine as a standard for comparison, the binding affinities of various tricyclics and other potent sodium channel blockers will be determined in voltage clamp studies on HEK cells transiently transfected with rat skeletal muscle and human heart sodium channel clones; native neuronal sodium channels in rat pituitary GH3 cells will also be used. Elements to be characterized include use-dependence of block and IC50 for resting and inactivated channel block. The working hypothesis for this phase of the studies is that duration of block in vivo will correlate positively with use dependence and negatively with IC50 for inactivated channel states. The in vivo studies will employ behavioral endpoints to examine both sensory and motor nerve block of sciatic nerve following a single injection of each agent in rats; drugs effective in rats will also be tested in the cauda equina space in sheep to model spinal routes of therapy. Drug design and synthesis will initially employ amitryptiline derivatives. Studies of the receptor site in the sodium channel will probe for the locations of two hydrophobic domains using point mutations and studies of drug potency on the mutated channels in HEK cells, with a special emphasis on residues which may be responsible for high-affinity binding of the tricyclic ring. Eventually the studies will be extended to other classes of drugs including phenylacetamides, calcium channel blockers, and a potassium channel blocker that also potently blocks sodium channels. Like tricyclic antidepressants, these agents have multiple phenyl rings but they are separated into two large hydrophobic domains rather than one.